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Isotopes in Environmental and Health Studies
ISSN: 1025-6016 (Print) 1477-2639 (Online) Journal homepage: http://www.tandfonline.com/loi/gieh20
Comparative equilibration and online technique
for determination of non-exchangeable hydrogen
of keratins for use in animal migration studies
L. I. Wassenaar & K. A. Hobson
To cite this article: L. I. Wassenaar & K. A. Hobson (2003) Comparative equilibration and
online technique for determination of non-exchangeable hydrogen of keratins for use in
animal migration studies, Isotopes in Environmental and Health Studies, 39:3, 211-217, DOI:
10.1080/1025601031000096781
To link to this article: https://doi.org/10.1080/1025601031000096781
Published online: 24 Sep 2010.
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Isotopes in Environmental and Health Studies
Vol. 39, No. 3, September 2003, pp. 211–217
COMPARATIVE EQUILIBRATION AND ONLINE
TECHNIQUE FOR DETERMINATION OF
NON-EXCHANGEABLE HYDROGEN OF KERATINS
FOR USE IN ANIMAL MIGRATION STUDIES
L. I. WASSENAAR
a,
* and K. A. HOBSON
b
a
Environment Canada, National Water Research Institute, 11 Innovation Blvd., Saskatoon, SK,
Canada, S7N 3H5;
b
Environment Canada, Canadian Wildlife Service, Saskatoon, SK, S7N 0X4
(Received 19 July 2002; In final form 14 November 2002)
Stable hydrogen-isotope ratios (dD) of keratin provide a novel means for tracking geographical movements of birds
and other species. Here we describe a rapid, low cost, analytical approach to facilitate online continuous-flow isotope-
ratio mass spectrometry (CF-IRMS) dDanalyses of keratins (120–160 samples per day) through the use of calibrated
keratin working standards and ‘‘comparative equilibration’’ to correct for the effects of moisture on exchangeable
hydrogen. It is anticipated that this analytical approach and CF-IRMS will greatly aid in providing cost effective
and directly comparable dDresults on keratins and feathers among various laboratories and researchers involved
in animal migration studies.
Keywords: Birds migration; Deuterium; Feathers; Hydrogen exchange; Hydrogen isotopes; Natural variations;
Standards
INTRODUCTION
The recent development of stable hydrogen isotope (dD) techniques to track migratory birds
and mammals represents a significant advance over traditional mark-recapture methods in
quantifying migratory connectivity [1, 2]. The stable hydrogen isotope tracking approach
exploits the fact that predictable continent-wide patterns of deuterium abundance in rainfall
occur on the continents and are transferred from plants through diet to upper trophic-level
consumers such as birds and insects. Hence, stable-hydrogen isotopic analysis of metaboli-
cally inactive tissues, like feathers, can yield quantitative geographical information on natal
origins of organisms [3,4]. Since these first successful demonstrations of the utility of dDin
determining breeding origins of birds and insects, this approach has rapidly gained accep-
tance, particularly in North America where subsequent studies have confirmed this relation-
ship and have applied the hydrogen isotope approach to a variety of novel avian migratory
questions [5–12]. Recently, interest has developed in applying this technique in Europe
where numerous avian species migrate to sub Saharan Africa [13]. Numerous stable isotope
* Corresponding author. Tel.: 306-975-5747; Fax: 306-975-5143; E-mail: len.wassenaar@ec.gc.ca
ISSN 1025-6016 print; ISSN 1477-2639 online #2003 Taylor & Francis Ltd
DOI: 10.1080=1025601031000096781
studies on migrating birds are currently underway across the US, Canada and Europe and
reports in the scientific literature are expected to increase significantly in the next few years.
Despite mounting interest in using dDmeasurements to track large-scale terrestrial animal
movement, several issues face researchers who wish to utilize this analytical tool to study
animal migration. These include isotope laboratory accessibility, analytical costs, and the
problem of uncontrolled hydrogen exchange in organic samples [14]. While the first two
impedances are overcome by adequate project budgets, the latter is not. It is well established
that dDmeasurements of feathers (and other organic materials) are problematical compared
to d
13
C and d
15
N analyses due to the problem of uncontrolled hydrogen isotopic exchange
between ‘‘exchangeable’’ organic hydrogen in the feathers with isotopically variable ambient
moisture in the laboratory environment. If left uncorrected, dDmeasurements of the total
hydrogen in an identical feather will yield different results at analytical laboratories located
at different geographic locations, as well as over time due to geographical and seasonal
changes in the hydrogen isotopic composition of ambient moisture [14]. The net result,
unfortunately, is incomparable concerning dDresults for feathers and other organic materials
both among labs and studies. As stable hydrogen isotope research in avian studies increases
and data begin to accumulate, it is imperative that keratin dDresults among labs and studies
be made comparable in order to link study results and to generalize our findings.
Previously, we described in detail an offline steam equilibration method for feathers and
organics that provided dDmeasurements that were not influenced by uncontrolled hydro-
gen isotope exchange with ambient moisture in the laboratory. This approach also yielded
isotopic values for the non-exchangeable hydrogen (dD
n
) of feathers [14]. This offline
method, however, required intensive preparations and dual-inlet hydrogen isotope analyses,
and hence suffered from low sample throughput (60 samples per week) and high cost
due to labor costs and preparative equipment required. While this approach was a step
forward in making dDresults comparable among avian studies, its adoption today requires
an adaptation to accommodate the most recent developments in hydrogen isotope analysis
of organic compounds by online continuous-flow isotope-ratio mass spectrometry
(CF-IRMS, [15]).
Here we describe a modification of our previous method of [14] to facilitate high-through-
put CF-IRMS keratin deuterium analyses (120–160 samples per day), and a ‘‘comparative
equilibration’’ approach through the use of calibrated keratin working standards. It is antici-
pated that this analytical approach and the use of CF-IRMS will greatly aid in providing
researchers with cost effective and directly comparable dDresults on feathers and other ker-
atin-based samples among various laboratories and studies.
MATERIALS AND METHODS
Keratin Standards
We prepared three in-house keratin standards (chicken feather (CFS), cow hoof (CHS), and
bowhead whale baleen (BWB-II)). The CFS was obtained from a single batch of chicken
feathers from a poultry processing operation located in Wynyard, Saskatchewan, Canada.
Approximately 2kg of feathers were obtained. The CHS (approximately 500 g) was obtained
by cutting hoof from a single cow carcass at an abattoir in Saskatoon, Canada. The BWB-II
was a powdered whale baleen obtained from Don Schell at the University of Alaska
(Fairbanks). Each keratin type was obtained from a single geographic location, and repre-
sented both terrestrial and marine end members in anticipation of obtaining a wide range
in dD
n
values.
212 L. I. WASSENAAR AND K. A. HOBSON
All of these keratin standards were solvent cleaned (2:1 chloroform:methanol solution),
dried, cryogenically ground, and homogenized using a mill in large enough quantities
(>0.5kg) sufficient to last several years in our laboratory The in-house keratin standards
were then analyzed 6 times each using the offline equilibration dual-inlet method described
by Wassenaar and Hobson (2000) and yielded the following results for dD
n
:
CFS¼1385‰ (VSMOW), CHS¼1872‰ (VSMOW), and BWB-II¼1084‰
(VSMOW). The dD
n
values reported for our in-house keratin standards include an assumed
equilibrium isotope fractionation factor of 80‰ between the steam (130 C) and the
exchangeable hydrogen in keratin (see details in 14). All of these keratin materials had simi-
lar chemical and exchangeable hydrogen (153%) properties as feathers.
A Comparative Equilibration Approach
Previous studies showed that hydrogen exchange between lab air moisture and exchangeable
hydrogen in keratins is fast, and will reach equilibrium within 24–48 hours at room tempera-
tures [5,16], and in less time at higher temperature [14]. Because the previous offline steam
equilibration approach was both costly and laborious and was required to be applied to all
samples, an alternative solution was to air equilibrate pre-calibrated keratin working stan-
dards (see above) and unknown feather samples with lab air moisture. The co-equilibrated
keratin standards and unknown feathers were then analysed in a single analysis session.
The benefit of comparative equilibration is that it eliminated the need for offline steam equi-
libration of samples and the associated costly equipment, thereby significantly reducing
analytical cost and processing time by not having to process samples offline. Further, this
approach follows the recommended Principle of Identical Treatment (PIT) for stable isotope
analyses, whereby samples and working standards are not only identical in their chemical
composition, but also go through exactly the same preparation and analysis steps [17].
The target weight of feather (and in-house keratin standard) required for routine online
hydrogen isotope analysis by CF-IRMS in our laboratory was 35010 mg. Feather samples
were cut (not powdered) from the same position on each feather and weighed using a micro-
balance (Sartorius M2P) and transferred into 4.0mm3.2 mm isotope grade silver capsules
(Elemental Microanalysis, Part #D2000). Capsules containing the keratin standard powder or
feather were folded into tiny balls. All weightswere recorded and the samples were stored in
96 position culture trays (Elisa plates), loosely covered with the lid. Samples and keratin stan-
dards were then allowed to ‘‘air equilibrate’’ on the shelf with ambient lab air moisture at
room temperature for >96 hours prior to stable hydrogen isotope analysis. The hydrogen
exchange process could be sped up by holding the samples and working standards in an
oven at higher temperatures since [14] showed that complete exchange with moisture occurs
in less than 30 minutes at temperatures above 100C. For practical purposes, however, a 96-
hour equilibration at room temperature was deemed sufficient and easier, and could be easily
implemented as a standard laboratory analysis procedure. Following 96-hour comparative
equilibration, all samples and standards were immediately loaded into the auto sampler car-
ousel of the CF-IRMS and analysed for dD, as described below.
Online Pyrolysis and CF-IRMS Measurements
Stable hydrogen isotope measurements on feathers and in-house keratin standards were per-
formed on H
2
derived from high-temperature flash pyrolysis and by CF-IRMS. Pure H
2
was used as the sample analysis gas and the isotopic reference gas. A Eurovector 3000
TM
high temperature elemental analyzer (EA) with auto sampler was used to automatically
pyrolyse feather samples to a single pulse of H
2
gas (and N
2
and CO gas). The pyrolysis
ONLINE ORGANIC DEUTERIUM ANALYSIS 213
columnconsistedofastandardceramictubewith an inner glassy carbon tube, which wasfilled
from the base to the heated zone with glassy carbon chips [see setup details in Ref. 15]. The
pyrolysis column was held at 1270C and the GC column at 100C. The GC column used to
resolvethesampleH
2
from N
2
and CO was a 1 meter 5 A
˚packed molecular sieve column. The
column flow rate was set to 100 mlHe=min and the sample purge rate to 50mlHe=min. The
resolved H
2
sample pulse was then introduced to the isotope ratio mass spectrometer
(Micromass Isoprime
TM
with electrostatic analyser) via an open split capillary.
The dDof sample hydrogen was calculated by measurement of HD isotopes at a m=zratio
of 3=2 (after standard H
3þ
corrections) and comparison to 2 reference pulses (10 nA and
5nA) of research grade (99.999%) H
2
gas having a dDof about 71‰ (VSMOW). The
IRMS source was empirically tuned and tested for optimum source linearity for H
2
at a
trap current of 400mA (0.5‰ per nA or V). The two 30-second reference H
2
pulses were
automatically introduced to the source using the dual-inlet reference and sample bellows.
Samples were automatically dropped into the Eurovector EA pyrolysis furnace 15 seconds
after initiation of the analysis run, with the sample H
2
pulse appearing in the IRMS at around
65 seconds. Single feather and keratin analysis time, after allowing for complete elution of
N
2
and CO from the GC column, was 165 seconds.
All feather and keratin dD
n
results are reported in units of per mil (‰) relative to the
VSMOW-SLAP standard scale. Repeated analyses of hydrogen isotope inter-comparison
material IAEA-CH-7 (100‰ VSMOW), routinely included as a check, yielded an external
repeatability of better than 1.5‰ for dD
n
.
RESULTS AND DISCUSSION
A typical example dDanalysis of keratin standards and unknown migrant bird feathers are
shown in Table I. The uncorrected hydrogen isotope values of the keratin standards from
each CF-IRMS auto run (2.5 hours for 50 samples) were subjected to a least squares regres-
sion to derive a correction formula to be applied to all the keratin standards and feather sam-
ples within that auto run (Fig. 1). A typical auto run included our in-house keratin standards
at the beginning, 2 keratin standards after every 10–12 unknowns, and 2 or 3 keratin stan-
dards at the end of the auto run.
A further check on the quality of online dDanalysis was to continuously monitor and
record sample H
2
peak height and H
2
sample peak retention times, as shown in Table I.
Peak height outliers sometimes occurred due to human sample weighing errors. Because
of slight IRMS source non-linearity any samples weighed to 350 10mg that yielded
peak heights greater or less than 1nA (Tab. I) of the mean were discarded and repeated.
In Table I this would correspond to a peak height of 9.5 nA, and so any samples with a
peak height less than 8.5nA and greater than 10.5nA would be discarded. This criterion
can be determined experimentally for each specific CF-IRMS instrument’s source linearity.
Alternately, because sample peak height correlates strongly with peak area, this parameter
could also be routinely monitored as a quantitative and comparative measure of yield.
Of course, sample peak height is also a function of the H content of the sample, and so any
minor variations in H content due to sample matrix or feather type were accommodated for
by either adjusting the weight of the keratin standards or the weight of the samples to ensure
an equivalent yield of H
2
gas by pyrolysis. To avoid unneeded complexity, we do not recom-
mend mixing of different sample and standard types within a single auto run, as the PIT also
recommends [16].
After 180 samples, accumulation of Ag from the sample capsules will begin to induce
column plugging. The symptom of column plugging is a shift in sample peak retention
214 L. I. WASSENAAR AND K. A. HOBSON
time and concomitant drop in sample peak height (Tab. I), as well as variable results. Prior to
reaching this point, removal and cleaning of the pyrolysis furnace column is required. The
use of larger Ag capsules (e.g. 46mm) resulted in earlier constriction of the columns,
after 90 samples. Rejuvenation of the pyrolysis column simply required cooling and
removal of the ceramic and glassy carbon tube, removal of the silver plug (it rolls out)
and the glassy carbon chips from the carbon tube, sieving and cleaning of the chips to remove
ash, and reassembly of the column as per manufacturer specifications. Our experience has
shown that a single ceramic and glassy carbon tube combination will last for years, but
we replaced our glassy carbon chips after about 2000 samples, based on visual inspection.
Frequently, 150mg samples of IAEA CH-7 polyethylene intercomparison material (having
no exchangeable H) and a value of 100‰ (VSMOW) are included in several sample auto
runs each week to monitor instrument performance and also to observe the apparent effect of
changes in ambient lab moisture on our keratin standards. Over a 3-month period in the
spring of 2002 we observed a maximum variation of 25‰ in a single keratin working stan-
dard relative to CH-7 simply due to effects of seasonal changes in the isotopic composition of
lab air moisture on exchangeable H.
In 2002, the cost of online keratin dDanalyses ranges from $20–35 USD per sample. As
more laboratories make this type of dDanalysis available the costs will likely decrease.
Considering the cost of dual inlet organic dDanalyses (>$100) or, more importantly, the
huge cost and efforts involved in largely ineffective conventional mark-recapture programs,
it is clear that stable hydrogen isotope analyses heralds an effective and highly affordable
means to begin to unravel migratory connectivity issues for many migrating species.
TABLE I Selected dDResults From an Auto Run of 35010mg Feather Samples and All Keratin
Standards (Boldface). Data Evaluation Includes Examination of Sample Retention Time (RT) and Peak
Height. Final Recalculation of dD
n
(VSMOW) is Based on a Least Squares Regression of the Known
Keratin Standards as in Fig. 1. Sample 39 is an Outlier*, Likely Due to Weighing Error, and So Was
Discarded in the Correction Due to Our Peak Height Criteria.
Sample
number Sample
name Peak retention
time (Sec) Peak
height (nA) dD
(uncorrected) dD
n
(VSMOW)
1CFS 64.6 9.9 58.1 141.0
2CFS 64.7 10.3 59.4 142.6
3CHS 64.7 9.8 92.8 184.7
4CHS 64.6 10.3 95.4 188.0
5BWB 64.6 10.0 30.1 105.7
6BWB 64.5 10.2 29.9 105.5
7 f1 64.7 9.3 21.7 95.1
8 f2 64.8 8.9 7.1 76.7
9 f3 64.6 9.5 35.6 112.7
20 g2 64.7 9.8 13.4 84.7
21 g3 64.7 9.5 17.9 90.4
22 g4 64.9 10.1 22.5 96.2
23 CFS 64.7 9.9 58.6 141.6
24 CHS 65.0 10.0 91.5 183.1
25 BWB 64.7 9.5 30.7 106.5
26 g5 64.7 9.4 21.6 95.0
27 g6 64.7 9.6 15.9 87.8
35 h2 65.2 9.2 19.7 92.6
36 h3 65.1 9.5 19.6 92.5
37 h4 65.0 8.5 16.1 88.1
38 CFS 65.1 9.8 57.9 140.8
39 CHS*65.1 6.7 89.2 *
40 BWB 65.3 9.3 31.5 107.5
Note: Mean CFS¼58.5, Stdev¼0.7, n¼4; Mean CHS ¼93.2, Stdev¼2.0, n¼3; Mean BWB ¼30.6,
Stdev¼0.7, n¼4.
ONLINE ORGANIC DEUTERIUM ANALYSIS 215
SUMMARY
Here we describe howonline keratin dDanalyses by CF-IRMS technology and the introduc-
tion of keratin standards and comparative equilibration can be used to provide cost effective
and rapid dDanalyses for large-scale animal migration research programs. We strongly
recommend that researchers utilizing organic dDanalyses adopt this protocol in order to pro-
vide findings that are comparable among laboratories. The development and distribution of
feather and keratin working standards that have awide range of dD
n
can be done on an indi-
vidual basis, or preferably, developed and distributed as a future collaborative effort among
avian researchers and stable isotope laboratories that provide feather dDanalyses.
Acknowledgements
We thank Patricia Healy for assistance in preparing and weighing feather samples for stable
isotope analysis. Funding was provided by Environment Canada.
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